Cholangiocarcinoma (CCA) is a lethal malignancy with features of biliary tract differentiation with limited therapeutic options. Thus, the long-ter goals of this program are to understand the pathogenesis of CCA and develop new therapies for its treatment. The current overall objective is to explore the importance of cholangiocyte deciliation, a potential pathologic process recently discovered by us, in the pathogenesis and progression of CCA. The CENTRAL HYPOTHESIS is that in CCA, primary cilia resorption resulting from exportin-5/miRNA/HDAC6 dysregulation disrupts the normal, ciliary dependent, tumor-suppressor signals that protect cholangiocytes from malignant transformation. Specific Aim #1 will characterize the mechanisms of ciliary loss in CCA by testing the hypothesis that the dysregulation of the nuclear-to-cytoplamic transporter, exportin-5, results in a decrease in tumor suppressing miRNAs, such as mir-433 and mir-22, inducing the upregulation of HDAC6 protein levels and ciliary loss. Specific Aim #2 will assess the consequences of ciliary loss by testing th hypothesis that promotes the disengagement of the normal, ciliary-dependent, tumor- suppressor external cues resulting in persistent MAPK and Hedgehog signaling, p53 downregulation, and cell growth. Finally, in Specific Aim #3 we will evaluate the outcomes of cilia rescue in CCA animal models. Our working hypothesis is that the restoration of primary cilia by targeting HDAC6 (shRNAS or pharmacologically), or mir433 and mir-22 (miR-mimetics), ameliorates tumor progression by rescuing the cholangiocyte ability to transmit ciliary dependent tumor suppressor external cues. The approach is innovative because it proposes to utilize the signaling pathways induced by the sensory functions of primary cilia in cholangiocytes as a tumor suppressive mechanism. Cilia have only recently become the subject of intense investigation, and since ciliogenesis is potentially reversible, studies directed at cilia are important. The proposed research is significant because it is imperative to identify the cellular networks regulating disease initiation and progression in order to develop better therapies. The results of the present proposal will uncover novel and generalizable information on fundamental, ciliary-dependent mechanisms controlling the proliferation of malignant cells and provide the foundation for plausible, novel anti-cancer therapies based on the restoration of primary cilia integrity.